TW201106986A - Novel compound from origanum vulgare and use thereof - Google Patents

Abstract

The present invention relates to a composition for whitening or antiaging, which comprises a compound of formula wherein R1 is hydroxy or C1-6 alkyloxy; R2 is hydroxy or C1-6 alkyloxy; provided that R1 and R2 are not hydroxy at the same time.

Description

201106986 VI. Description of the Invention: [Technical Field] The present invention is a novel compound derived from oregano (βτ/Paiwan (10) and its use [Prior Art] In the past few years, it has been applied to traditional herbs and medicinal plants. The development of new medicines and health care products is increasing day by day. These days have been proven to be anti-oxidant, anti-inflammatory and anti-high Φ blood pressure and other related drugs. Plant derivatives products are gradually accepted and adopted in the cosmetics industry. Therefore, it is worthwhile to develop other alternative food additives or cosmetic products by extracting the natural ingredients obtained from medicinal plants, herbs and spices. ~M〇ri卿um vuigare) About Oregano (〇々_ The type of cockroach and its application have been found to be antibacterial and antioxidants. Oregano (5). Foot _ order) is an aromatic plant, which is used as a spice in the Mediterranean region. Previous studies have confirmed that the main efficacy of oregano containing good essential oil ingredients should be called for antibacterial aspects. However, there is very little research on the mechanism of oregano whitening and antioxidants. Whitening mammals t, melanin stimulating hormone (a_MSH) is a necessary condition for pigmentation. LH binds to melanin receptor i (MC1R), which inhibits cyclosporin gland) B and activates small eye deformity-related transcription factors. (microphthalmia-associated transcription fact〇r, referred to as bribe) causes melanin production. The upstream regulation of black f-enzymes, such as __, amines, can cause undesirable browning in fruits and vegetables and produce an indispensable enzyme in melanin in the skin, hair and eyes of animals. Lactase plays a part in the initial melanin synthesis process involved in the oxidation of dopa by L-Lalase and the conversion of 201106986 dopa to dopaquinone. Dopaquin is automatically converted to dopachrome. Tyrosine-related protein-2 (TRP-2) / dopachrome tautomerase (TRP2/DCT) catalyzes the conversion of dopachrome to 5,6-dihydroxyindole_2_carboxylate acid

(5, 6-dihydroxyindole-2-carboxylic acid, abbreviated as DHICA). Tyrosine-related protein-1 (TRP-1) (TRP1/DHICA oxidase) catalyzes the oxidation of DHICA to form 吲哚-5,6- stupidin-2-acidate (indole-5,6-quinone-2-carboxylic Acid). The two closely related structures of TRP2/DCT and TRP-1 produce unstable quinines, which are further aggregated to produce melanin. Pigmentation of the epidermis and dermis depends on the increase in the number of melanocytes or the activity of genes involved in melanin production and enzyme activity. In the case of ultraviolet light, chronic inflammation and dermatitis, alpha-MSH release triggers pigmentation. In recent years, many natural and synthetic substances have inhibited tyrosinase activity and thus can be used as a skin whitening agent, but there is still much controversy about the safety of the skin. Previous studies have indicated that several currently known tyrosinase inhibitors, including arbutin, tannic acid and linseed oleic acid, have been widely used in skin whitening but have a side effect. Therefore, the safe concentration must be determined before the skin whitening test. An free radical contains one or more atoms, molecules, or ions of unpaired electrons. Most of the chemical properties are in a highly unstable state. The reactivity is higher than that of other molecules, and it will grab the electrons of the neighboring molecules to form electrons. Yes, the state that is robbed of electrons will become unstable, and if it is highly active, it will cause a chain reaction to promote the generation of free radicals. When free radicals attack giant molecules inside or outside the cell, such as lipids, proteins or nucleic acids, the membrane integrity is easily impaired by lipid oxidation on the membrane; the amino acid residues of the protein are oxidatively modified, such as several groups. The formation of (carb〇nyl) makes important enzymes inactive; when DNA is attacked by free radicals, it causes DNA base modification, cross-linking (cr〇ss_link), DNA single-strand (or double-strand) breakage, etc. And cause a lot of damage to the gene, and lead to abnormal cell function, which in turn affects the health of the entire 201106986 object. Many diseases such as atherosclerosis, complications of diabetes, amyotrophic lateral sclerosis, neuropathy, autoimmune diseases, and aging are all associated with oxidative damage. Among them, diseases such as atherosclerosis caused by abundant materials have become a serious problem for Chinese people. Some scholars have pointed out that the oxidation of low-density lipoprotein (LDL) has a great correlation with the incidence of this disease. The most important method to prevent oxidation is to remove oxygen, and secondly to remove the promoter of auto-oxidation. Therefore, the so-called anti-xidant is any substance that can delay the oxidation of fat, or the oxidation of other substances. As far as the living body is concerned, it can protect the biological matrix against antioxidants, whether in the food system or in the biological system, the presence or absence of antioxidants is the most important way to prevent lipid Weihua, and the ideal antioxidant ship The following conditions are available: (a) Low concentration is effective. (b) Wide applicability. (c) No bad taste or color. (d) Not toxic. (e) Low prices. Antioxidants can be classified into free radicals (free radicai terminat〇s), reducing agents (reducants) or oxygen scavengers, metal chelators (fat plus chelators), enzyme types. Enzymic anti〇xidants and peroxide decomposers. Free radical terminators, also known as primary antioxidants, provide antioxidants by providing hydrogen atoms, which provide the primary antioxidant effect, most of which are antioxidants. It is a phenolic compound, such as propyl gaiiate (pg), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BUT) and tributyl hydrogen (tertiary-butyl hydroquinone 'TBHQ for short). The antioxidant mechanism of the reducing agent or oxygen scavenger is to transfer wind atoms or scavenge oxygen molecules, such as α-tocopherol, which has a faster reaction rate with peroxide radicals than lipid radicals. And achieve the role of anti-oxidation. Chelating agents or co-oxidants These substances do not have antioxidant properties themselves, but because of their unshared pain of electrons, they can combine with the 201106986 oxidized transition metals (Fe2+ and Cu2+) to form stable compounds. Inhibition of oil self-oxidation reaction 'such substances can be represented by citric acid, polyphosphates, diamine hypothyltetraacetic acid (EDTA) and its salts. The representative of the peroxide decomposing agent is dilauryl thi〇pr〇pi〇nate and thiodipropionic acid, and its mechanism of action is to decompose the lipid peroxide into the final product of diazepam. . In recent years, many studies have explored plants with antioxidant capacity and their activities as a basis for the selection of antioxidant foods. SUMMARY OF THE INVENTION The present invention provides a compound having the formula

Ri

R2 Bu. -Η 〇 wherein R! is a hydroxyl group or c,·6 alkoxy group; R2 is a hydroxyl group or a Ci 6 alkoxy group: but % or % are not a trans group. Among the compounds of the present invention, preferred R! is a hydroxyl group; preferably R2 is a methoxy group. Therefore, the best compound of the present invention is

H 1C —Η ο

3 Η C ο 6 201106986 The invention also provides compositions comprising a compound of the invention. The composition of the present invention has whitening and anti-oxidation ability and can be applied to skin of whitening or anti-aging individuals, and can also be used as a raw material for cosmetics, skin care products or health foods. Individuals receiving the compositions of the invention include, but are not limited to, mammals. In a preferred embodiment of the invention, the compositions of the invention have the ability to inhibit melanin production. In a preferred embodiment of the invention, the composition of the invention inhibits protein expression of the small eye deformity related transcription factor (MITF), tyrosinase or tyrosine related protein 2 (TRp_2). In a preferred embodiment of the invention, the composition of the invention has the ability to scavenge free radicals. In a preferred embodiment of the invention, the free radicals are free radicals and free radicals. In a preferred embodiment of the invention, the compositions of the invention reduce the levels of intracellular reactive oxygen species (ROSs) and have the ability to inhibit lipid peroxidation. The words "including", "having", "including", or "including" as used in the specification and the scope of the patent are included in the (10) or the syllabary. Surgery-inhibition, "reduction" or "prevention" or any variant of these terms includes any measurable reduction or complete suppression to achieve the desired result when applied to a patent and/or book towel. The term "effective" as used in the specification and/or patent application is intended to be sufficient to achieve the desired, desired or expected result. 201106986 [Embodiment] The present invention may be embodied in different forms and is not limited to the examples mentioned in the following essay. The following examples are merely representative of the various aspects and features of the present invention. Example 1 Material 舆 Method Test material Oregano (β-eye from the traditional Chinese medicine shop, after the sample was verified by Professor Ding Xiuyu, the dried samples were stored at Jianan University of Pharmacy and Technology. Tyrosinase, levodopa (L-D0PA), Arbutin and vitamins were purchased from Sigma Chemical Co., St. Louis, USA.

The melting point was measured using a melting point meter (MEL-TEMP 50/60 CYCLES, 110-120 volts - 200 watts). The optical rotation was measured using a polarimeter (SCHMIDTHAENSCH, p〇LARTR〇NIC _). UV spectroscopy was obtained using a spectrophotometer (shimadzu, UV-160), and infrared spectroscopy was obtained using a spectrometer (ThermoMattson, IR 300). Nuclear magnetic resonance hydrogen spectroscopy was recorded at 500 MHz using a liquid superconducting nuclear magnetic resonance spectrometer (BRUKER AVANCE 500 Solution-NMR spectrometer). The carbon spectrum was obtained using a liquid superconducting nuclear magnetic resonance spectrometer (BRUKER AVANCE 500 Solution-NMR spectrometer at 125 MHz in CDaOD). Fast atomic impact mass spectrometry (FABMS) was obtained by mass spectrometer (JEOL TMSD-100) analysis. High performance liquid chromatography (Shim-pack PREP-pheny 1 co 1 umn, Shimaduz Corporation) utilizes reverse phase chromatography. Example 2 Purification, isolation and structural identification of origanoside 201106986 Weighing oregano 10.0 kg The medicinal material was ground to a fine powder and extracted with 95% ethanol to obtain an extract of 930·0 g. The extract is subjected to systematic partition extraction using solvents of different polarities. First, a mixture of n-hexane and 95% methanol was used to obtain a n-hexane layer (e.g., g). The 95% methanol layer was concentrated to remove methanol, and then subjected to partition extraction with ethyl acetate and water to obtain ethyl acetate layer (195.0 g) and water layer (835.0 g). The ethyl acetate layer was purified by molecular sieve (LH-20) using methanol as an extract to obtain four fractions. The third part is prepared by preparative low pressure reverse phase chromatography column (L〇barRP-8), using solvent methanol and purified water (5〇:5〇) as the extract for purification and separation, followed by high pressure preparation. A liquid chromatograph (HpLC) was purified by methanol and purified water (95:5) to obtain a novel phenolic compound (5 〇·7 mg), and the name was named origanoside. Oreganoside is an orange solid; the melting point is 140-141 degrees; the optical rotation is -160.5. (c=0.81, MeOH); UV absorption values were 208, 212, 262 and 292 nm. Infrared absorption was 3403, 1636 cm-1; high resolution mass spectrometer (HRFAB-MS) measured 436.1350, The molecular formula is C2 crystal 4〇1(); the molecular weight measured by FAB mass spectrometer is 436; the data measured by 1H and 13C NMR of the nuclear magnetic resonance apparatus are shown in Table 1. The oreganoside compound is confirmed by various spectral analyses. As shown in Figure 1. 201106986 Table 1. Hydrogen and 竣 spectral data of oxoside (cd3〇d) Position sc 5H(7inHz) 6 12 1'2'3'4' 6, H3 C ο 124.1 117.2 7.43 (2.0), d 147.6 1S3 .6 112.0 6.97 (8.5), d 123.6 7.54 (8.5, 2.0); dd 168.0 159.2 117.9 7.11 (8.5), d 130.9 7.38 (8.5), d 131.8 130.9 7.38 (8.5), d 117.9 7.11 (8.5), d 67.3 5.24, s 102.3 4.92 (5.0), d 71.5 3.45 (SO), t 78.1 3.46 (5 0). t 75.1 3.93 (5.0), t 78.3 3.43 (5.0,2.〇Xdd 62.7 3.69 (12.0,2.0), d 3.87 (12.0), dd 56.6 3.90, s Example 3 Cell Culture and Assay Human fibroblasts (Hs68) and mouse melanoma cells (B16F0) from the American Type Culture Collection (Rock-ville, Maryland) ) Cell culture in DMEM (Dulbecco, s Modified Eagle Medium medium) medium (GIBCO, Grand Island, NY), 10% fetal bovine serum (Hazelton Product, Denver, PA, USA) after adding 1% penicillin-streptomycin , Incubate in a 5% C〇2 '37 ° C incubator. Dissolve the reagent in 1% DMS0 at a concentration of 1〇〇mg/ml. Add the desired concentration in the culture solution. The final concentration of DMS0 is not more than 0.1%. Cell viability test 201106986 The number of cells of 100/d 1.5χ105 cells/ml is cultured in a 96-well culture plate (96_weU plate) at 37 C , growing in a 5% C〇2 incubator for more than 24 hours. Adding different concentrations of oreganosides after 72 hours, using (3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyl -tetrazolium bromide) test to measure its viability. After the cells are cultured for a certain period of time, add the solution (0.5? / 1111) for four hours, then add 2 () 0; ^ 11) 1 ^ 〇 dissolve the precipitate (Formazan) The absorbance was measured at a wavelength of 570 nm (BioTek, SynergyTM 2, USA). Different concentrations of samples (0, 5, 10, 20, 50, and 100 #g/ml) were added to B16 and Hs68 cells by MTT assay to determine the safe concentration of oreganoside. The results are shown in Figure 2. The ornithine is not toxic at 0 to 100 //g/ml, and the survival of Hs68 cells is increased at high doses of 1〇〇. According to the early screening of whitening products, the concentration was based on the results of the results. The results showed that oreganoside was not toxic, and the subsequent tests were conducted to inhibit tyrosinase at two concentrations of 1 〇 and 20//g/ml. Elbow 811 (3-11161:117 Bu & 62〇1:11丨32〇1 to 0 fat 1^ buckle & 20116) Activity test Doxamase test for dopa (D0PA) oxidase activity using colorimetric assay When levodopa becomes DOPAquinone, dopaquinone reacts with MBTH to form a pink color with absorbance at 508 nm. B16 cells of 1 ml of 1.5 x 105 cells/ml of cells were cultured in 24-well multidishes and grown in a 37 ° C, 5% C 2 incubator for 24 hours or more. After adding α-MSH and different concentrations of oreganoside, after 72 hours, the medium was removed, washed with PBS, and then added with 1% surfactant (Triton X-100) to break the cells after freezing and thawing (-80°) After 30 minutes of C, 25eC for 25 minutes, and 37°C for 5 minutes), a mixture of 190 and 1 MBTH (10 mM MBTH, 1.1 mM levodopa, 4% dimethylformamide) was added and reacted at 37 ° C for 4 hours. The absorbance was measured at 508 nm (BioTek, Synergyra 2, USA). 11 201106986 Intracellular tyrosinase assay The number of cells in 1 ml 1.5x105 cells/ml was cultured in 24-well multidishes' and grown in a 37 ° C, 5% C 2 incubator 24 More than an hour. After adding α-MSH and different concentrations of oreganoside, after 72 hours, the medium was removed, washed with pbs, and then added with 1% surfactant (Triton X-100) to break the cells in a cold-beam and post-twisting manner (- After 80 ° C for 30 minutes, 25 ° C for 25 minutes, and 37 ° C for 5 minutes), 1 〇〇 y 1 10 mM levodopa was added and reacted at 37 ° C for 4 hours to measure the absorbance at 475 nm (BioTek, Synergy TM2, USA) The ability of geraniol to inhibit tyrosinase activity The involvement of tyrosinase affects the rate of melanin synthesis. The regulation of pigment formation depends on inhibiting or stimulating the activity of tyrosinase. Therefore, dermatologists and cosmetics manufacturers can improve the treatment of pigmentation in patients by inhibiting the activity of tyrosinase. After the effect of 10 or 20 μg/ml of oreganoside, arbutin and vitamin C on B16 cells for 72 hours, the results showed that the inhibition rate of ornithine to tyrosinase of b16 cells was close to 16.9. The inhibition rate of the sputum test is about 39. 4 to 59.2%. Oreganoside In the MBTH assay, inhibition of dopazyme activity showed a drug-dependent increase (丨〇 and 2〇//g/ml) (Fig. 3B). When oreganoside was compared with arbutin and vitamin C, the results showed that oreganoside was quite effective in inhibiting dopazyme. These results demonstrate that ornithine inhibits melanin synthesis primarily by reducing the activity of intracellular dopazyme. Quantitative analysis of MCIR, MITF, guanamine, TRP-1 and TRP-2 1. 5x 1〇5 cells/mi of B16 cells 'added to sun (丨yM) and 2〇"g/ml of oregano After 72 hours, 'MC1R, MITF, glutaminase, TRP-1 and TRP-2 antibodies were added (disposed in PBS at a ratio of 1 to 300). 〇 5% BSA (pBS_BSA) and 〇·1% sodium azide (sodiumazide) were added and dyed at 4 ° C for 45 minutes. After washing twice with iced PBS, it was incubated with Luguang isothiocyanate (FITC) in combination with an anti-mouse thief at 12 201106986 at 4 ° C for 30 minutes. The cells were washed with iced PBS and fixed with 4% paraformaldehyde. The nuclei were stained with the fluorescent dye Hearst 33342 (Hoechst 33342). The amount of MC1R, MITF, tyrosinase, TRP-1 and TRP-2 was observed using a fluorescent immunostaining experiment. The nucleus was obtained using a Multi-Detection Microplate Reader and analyzed by Gene5TM software. When the cells are treated with drug concentration, the percentage of fluorescence is the intensity of the FITC intensity versus the intensity of the fluorescent dye.

Immunofluorescence assay and flow cytometry were used to evaluate the expression of MITF, tyrosinase and TRP-2. The melanin synthesis-related protein' contained MTF, tyrosinase and TRP-2 in B16 cells, and sequentially added a. -MSH (1 //M) treatment of 20/zg/ml oreganoside, assessed by immunofluorescence staining and flow cytometry. After 72 h of oreganoside culture, the cells were treated with mice against MITF, tyrosinase and TRP-2 (antibody was placed in PBS at a ratio of 1 to 300). Add 0.5% BSA (PBS-BSA) and 0.1% sodium azide and stain for 1 hour at 4 °C. The cells were washed with ice-cold PBS and incubated with FITC-conjugated anti-mouse IgG (1 to 500) for 30 minutes at 4 °C. The nucleus was stained with a 萤/zg/ml fluorescent dye Hearst 33342 (H〇echst φ 33342) and then placed under a fluorescent microscope. This was done before the flow cytometry analysis of the cells, adding two 1 BPS wash steps using ice. Data is analyzed using software (WinMDI). Oreganoside regulation affects tyrosinase-related protein expression Melanin synthesis initiates activation of MITF by multiple pathways, in mammals, a_MSH binds to MCIR, increases cAMP, and activates MITF. MITF is a potent factor for efficient transcriptional activation of tyrosinase and TRP-2 and TRP-1 expression is considered to be a key regulator of melanocyte growth. In order to determine whether the molecular mechanism of oreganoside is blocked by melanin production in B16 cells, after 2 hours wg/ml of oreganoside is applied to B16 cells for 72 hours, immunofluorescence 13 201106986 light staining is used to quantitatively analyze MC1R, The expression levels and DNA content of MITF, lysine, trp-2 and TRP-1. As shown in Fig. 4A, the expression levels of MITF, tyrosinase and TRP-2 in bovine glycosides-treated B16 cells were significantly lower than those in the control group. The performance of MC1R and TRP-1 did not change significantly compared with the control group. After the effect of oregano on B16, the DNA content did not decrease' and the nucleus did not change (Fig. 4B). As confirmed by the cell viability test, oreganoside did not cause cell cytotoxicity. Furthermore, flow cytometry analysis confirmed that the amount of MITF and TRP-2 was decreased after 20/zg/ml of oreganoside was applied to B16 cells (Fig. 4C). The results showed that oreganoside reduced the expression of mitf, tyrosinase and TRP-2, thereby reducing melanin production. Example 4 Animal Test Preparation of a gel containing oreganoside Carbopol® 940 (Carbopol® 940' is a polyacrylic acid polymer) was donated by Lubrizol Advanced Materials, Inc., USA. The oreganoside gel was prepared by dispersing 1% carbomer 940 in the water of the mixture. The oreganoside at a concentration of 0.02% w/w was added to a normal physiological saline solution (0.9% NaCl (aq)) together with carbomer 940 powder, followed by magnetic stirring for 6 hours. 5。 The 〇. 。 1% triethanolamine (triethanolamine) to accelerate the dispersion rate, and neutralized to a pH of 7.4. The whitening effect of the animal's application of oreganoside clots The five-week-old C57BL/6J strain of mice, weighing about 20-50 grams, was obtained from the National Animal Laboratory of Taibei, Taiwan. Throughout the experiment, the rats were placed in stainless steel cages at a temperature of 25 ± 2 ° C and a day and night cycle of 12 hours. The mice had a seven-day acclimation period before the experiment. After the mice were depilated, after a day of rest, each mouse was divided into two parts along the vertebrae, and the right side was coated with a 1 g gel sample (control group) and the left side was coated with a gel sample containing oreganoside (experimental group) ). Twelve mice were smeared daily and the colorimetric values of the 201106986 gel sample and the oreganoside gel sample were determined. A color difference meter (DermaScan High Frequency Ultrasound, DSM II Colormeter, Crypro LN2, DermaLab and Dermaspectrometer, Cortex Technology) was used to evaluate the absorbance. In this standard instrument set 'contains: (WLED optimized), (red spot & melanin) and RGB values. The Z* value represents the colorimetric value, and the $ represents the degree of color expression on the red/green axis, representing the degree of color expression on the yellow/blue axis. According to the RGB value, quantify the erythema (magic and melanin (the pigment distribution of the fish. The E/m before and after the test is measured by the color difference meter). The color change of the skin can be confirmed by the colorimeter measurement. The erythema and the erythema are black (and must The skin color of 12 mice was quantified. As shown in Table 2, after applying the oreganoside gel for 10 days and comparing the coated gel sample (control group), the mice coated with oreganoside gel increased π, a wood decreased slightly and 汾There was no significant change. After the application of oreganoside gel, the erythema value (magic and melanin values were significantly lower than that of the control group and the uncoated gel. The results showed that oreganoside gel can make the skin brighter and inhibit The production of erythema and melanin. Therefore, oreganoside can be used as a new raw material for skin whitening cosmetic products. Table 2, comparison of smeared ten days of oregano glycosidic and unsmeared and i1/# value •-

L* a* b* E Μ Before, after, before, after, before, after, before and after the control group 3Π+0.8 32.4±1.1 7.8+2.1 7.3+1.7 2.0+0.8 0.8+0.7 6.1+0.5 5.3+1.1 45.9+0.7 44.5 +1.0 Experimental group 31.8+1.2 36.5+0.9 7.9+1.5 7.0+Π 1.2+1.0 U Soil 1.2 6.010.6 4.510.7 44.9+0.8 40J±U Each mouse is divided into two parts along the vertebrae, and only the gel sample is applied to the right side. (Control group), on the left side, a gel sample (experimental group) containing oreganoside was applied (n=12). Example 5 Evaluation of antioxidant capacity of oreganosides DPPH free radical scavenging efficiency 15 201106986 Samples of different concentrations of extracts were prepared, and 0.01 Μ sodium phosphate buffer solution (pH 7.4, 0-15 M NaCl) and 500 were sequentially added. # 1 Freshly configured 250/zM DPPH Ethanol solution was shaken and allowed to stand in the dark room for 20 minutes, and the absorbance at 517 nm was measured with a spectrophotometer. The lower the absorbance value, the stronger the ability of the sample to scavenge DPPH free radicals. The percentage of scavenging effect is obtained by [1-(the absorbance of the sample at 517 nrn/the absorbance of the control group at 517 nm)] x 100%. (scavenging effects %). Determination of ABTS free radical scavenging efficiency

Take different concentrations of the extract sample 'sequentially added 〇. 〇1 sodium citrate buffer solution (pH 7. 4, 0·15 M NaCl) buffer solution and 500 to 1 〇. 175 mM ABTS solution, after shaking in dark After standing for 10 minutes, the absorbance at 734 nm was measured with a spectrophotometer. The more the absorbance value is, the stronger the ability of the sample to remove ABTS free radicals is removed by [1-(sample absorbance at 734 nra/absorbance at 734 nm in the control group without added sample)] X 1〇〇% Scavenging effects %. The ability to correct the concentration of water-soluble vitamin E (trolox) to remove ABTS cationic free radicals was used as a standard curve. DPPH (2,2-Diphenyl-l-picryi hydrazyl) is one of the simplest anti-free radical methods. The main _ DPPiUb aged ethanol solution produces a stable free radical and produces a purple reaction. When the 胄DppH radical receives electrons to form an electron pair, its absorption peak at 5 Π nm will decrease or disappear. Therefore, it is judged whether or not the sample has the ability to supply a hydrogen atom to scavenge free radicals by measuring the change in the absorption value of 517 coffee. ABTS and the persulfate clock (p〇tassium_u cell & κ secret) produce an oxidation reaction to form a stable blue-green water-soluble • Cong + cationic free radical. · abts+ has absorption bands at 415, 645, 734, and 815 nm, but when ABTS+ is reduced by an antioxidant or combined with another radical, the kinetics will decrease or disappear. To test the sample to provide the ability to bribe _free radicals, so the lower the luminescence value, the stronger the ability to clear the ABTS free radicals. The antioxidant capacity was tested after adding various concentrations of oreganoside to 201106986 α, 5, 10, 15, 20 and 100 eg/ml). The results showed that oreganoside achieved a 76.87% inhibition rate at a high concentration of 100/zg/mi in the DPPH test, and a 98% inhibition rate in the ABTS cation scavenging free radical test, as shown in Figures 5 and 6. 0 The ability to inhibit lipid peroxidation is determined by the effect of test samples on lipid peroxidation. The microlipids are used as unsaturated fatty acids to mimic the cell membrane of the body. The added vitamin C can reduce iron ions (Fe, ferrous ions). Fe2+) 'catalyzes the lipid peroxidation of microlipids. At high temperature, thiobarbituric acid (TM) reacts with the maloxidized product malondialdehyde (MDA) to form a pink thiobarbituric acid reaction substance (TBARS). The absorbance measured at 532 nm shows the peroxidation. Take 10 liters of the liposome suspension 1.6 W and 〇·55 ^ of the ferrous sulfate, 〇. 55 y 1 of 10 mM of vitamin C, 75. 75 y 1 buffer solution and 25 " 1 different concentrations of compound were mixed separately, reacted at 37»c for one hour, then added 4〇/z 1 thiobarbituric acid reagent and 5.4 A 丨 〇丨M diamine hypoethyltetraacetic acid, heated at 100 ° C for 20 minutes, cooled After centrifugation at 12 rpm for 1 Torr, 1 〇〇1 supernatant was used to measure the malondialdehyde content at 532 nm. The results of the inhibition of lipid peroxidation of oregano lipids are shown in Fig. 7. It can be seen that the inhibition power of oregano is increased with the increase of concentration. At the highest concentration of 1 GMg/ml, the oregano inhibition rate is 61.4%, which is better than the vitamin c 49 _ inhibition rate. Oregano Wei Yuyun Xuan / Wei Zhuan (a) picked up _ Wei oxidation has a inhibitory effect, but has the effect of protecting the cell membrane.

17 201106986 Intracellular reactive oxygen species (R0S) content test using mouse hepatocyte cell line (BNLCL2) to add 1 v 1 oreganoside for 72 hours, washed with PBS, add fresh medium (DMEM), Add 1 y 1 〇·1遽 of peroxygen. Hydrogen 0. lmM 'After one hour, remove the culture solution, wash with PBS' and add fresh medium, add 1/zl 10/zM DCFH-DA ( After 2', 7'-Dichlorofluorescin diacetate) was applied at 37 ° C for one hour, the culture solution was removed, and quantitative pBS was added to measure the fluorescence value. When the cells are subjected to hydrogen peroxide, a series of reactions are performed to generate reactive oxygen species. The cells are stained with DCFH-DA, and the amount of active oxygen is known by flow cytometry to detect the scattered fluorescence. The experimental results show that compared with two well-known antioxidants, the ornithine is more effective than the two antioxidants in protecting the cells from reactive oxygen species (such as oregano). Figure 8). Thiobarbituric Acid Reactive Substance Test The thiobarbituric acid reactive substance is a product of reactive oxygen species that destroys lipid peroxidation induced by cell membrane lipids and is an indicator of oxidative damage of -4. When the free radical attacks the cell membrane, the lipid contains a large amount of unsaturated fatty acid, which causes lipid peroxidation and the product is malondialdehyde. It combines with two molecules of malondialdehyde to form a pink substance (TBA-MDA) in an acidic environment, and has a maximum absorption wave at 532 nm. This characteristic shows whether the sample has an effect of inhibiting the formation of malondialdehyde. After mixing 5 / zl tissue solution and 10 W of different concentrations of oregano, the mixture was uniformly mixed, and 20110086 was added 5 W of loo MM ferrous sulfate and strontium. After standing in the m incubator for one hour, 'add 25...m tri-acetic acid (trichl face cetic acid, abbreviated as gamma and 19-hour thiobarbituric acid in an 80T water bath for 20 minutes, 14,000 rpm for 1 minute) The absorbance of 532 ηιη was measured by spectrophotometer. The amount of C2 in the liver and brain tissue tissues of rats was observed at 20 and 100 (10) ml concentration of oregano, and the effect of inhibiting the oxidation of WEE was evaluated. The results are shown in Fig. 9's liver tissue fluid treated by Oregano, which inhibits the production of B. sinensis. 4 to 67. The brain tissue fluid that has been torn by oregano has been inhibited. 66. 9 to 83.4% 'especially the effect of oregano to inhibit the formation of malonate in brain tissue fluid than that of water-soluble vitamin E (trolox) (inhibition of 49.3 to 64 (10)). The above results indicate that oreganoside can effectively inhibit Lipid peroxidation. Statistical analysis called the mean value of the experimental knots. The statistically significant differences between the mineral samples and the unpaired samples were determined by independent and paired students. (5) Printing (5) Grabbing and paired Student' s i-test) to decide. When the control group is compared to more than one experimental group, the measurement is repeated using one-way variance or two-way variance. The Dannett check (〇unnett, s(4)) or the Student-Newman-Keuls test is performed whenever the 'AN0VA data differs'. Data analysis and image representation were performed using statistical plotting software (SigmaPlot software, Version 8.0, San Rafael, CA, USA). 201106986 More and more research shows that anti-oxidation is an important step in anti-aging. If it can eliminate excessive oxidative free radicals, it can prevent many diseases related to free radicals or aging. Through the above experiments, oreganoside has a good effect on the ability of ABTS and DPPH to scavenge free radicals, especially in the test of thiobarbituric acid reaction substance, which has a high inhibition rate for the cytosine produced in brain tissue fluid. In the in vivo reactive oxygen test, it can be known that Oregano has the ability to protect cells. These experiments have demonstrated that oreganoside has antioxidant capacity and can be developed into a natural antioxidant or health food in the future. Those skilled in the art will readily appreciate that the present invention can readily achieve the objectives and achieve the results and advantages mentioned, as well as those which are present therein. The cells & animals and their manufacturing procedures and methods in the present invention are representative of the preferred embodiments, which are exemplary and not limited to the field of the invention. Those skilled in the art will be aware of the modifications and other uses therein. These modifications are intended to be within the spirit of the invention and are defined in the scope of the patent application. The present invention has been described in detail with reference to the embodiments of the present invention, and the invention may be Spirit and scope. All patents and publications mentioned in the specification are subject to the general skill of the art in the field of the invention. All patents and publications are hereby incorporated to the same extent as if each individual publication is specifically and individually indicated. 201106986 = This invention illustrates the invention, may be in the absence of =_, the object is specific to a ^

1妣1 and expression 疋 as a description of the specification and not a limitation, and there is no intention to use the expression such as the wheel and the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ change. Therefore, it should be understood that although the present invention has been specifically disclosed in terms of preferred embodiments and any features, the present invention is still in the sufficiency of the present invention, and such modifications and variations are still in the invention. Inside. [Simple description of the diagram] Figure 1. Structure of the oregano bud (C21H24010) with a molecular weight of 436. Cell viability of B16 and Hs68 cells after oreganoside treatment. Using the test, the cell viability of Weili 72 small and fine sputum was expected. Figure 3 shows the ability to inhibit intracellular tyrosinase (A) and MBTH activity (B) after 72 hours of treatment with B16 cells with ore 10 and 20 "g/mi of oregano, arbutin and vitamin C. Figure 4. Protein expression of MC1R, MITF, tyrosinase, TRP-2 and TRP_1 after bovine glycosides were applied to B16 cells. The bovine glucoside at a concentration of 20/zg/ml was applied to B16 cells for 72 hours. The cells were washed with PBS and fixed with 4% paraformaldehyde. Nuclear staining was performed using Ginger dye Hearst 33342 (Hoechst 33342) (blue) and using a specific protein-bound fluorescent cursor to define FITC (green). (A) Protein expression of MC1R, MITF, tyrosinase, TRP-2 and TRP-1, quantitative analysis by immunofluorescence 4B) Observation of MC1R, MITF, tyrosinase, TRP under a fluorescence microscope (200x) Protein expression of -2 and TRP-1 21 201106986. (c) Analysis of protein expression levels of MITF, tyrosinase and TRP~2 by flow cytometry. Figure 5. DppH free radical scavenging ability of oreganoside. Figure 6. The free radical test of ornithine. Figure 7. The ability of oreganoside to inhibit lipid peroxidation of vesicles. . Figure 8. Content of intracellular reactive oxygen species (ROSs) after bovine glycosides. Figure 9. The ability of oreganoside to inhibit lipid peroxidation in rat liver tissue and brain tissue. Annex 1 is a color schematic of Figure 4B. [Main component symbol description] None.

Claims (1)

201106986 VII. Patent application scope: 1. A compound with the following formula Η 1C —Η ο 2 R wherein hydrazine is a hydroxyl group or a q_6 alkoxy group; r 2 is a hydroxyl group or a cN 6 alkoxy group; however, R 2 is not a hydroxyl group at the same time. 2. A compound according to item 1 of the scope of the patent application, wherein the hydroxy group is a sulphate. 3. A compound according to claim 1 wherein R2 is a methoxy group. 4. According to the compound of claim 1 of the patent scope, the system is 5. A composition comprising a compound of the formula Η 1C —Η ο R2 wherein & is hydroxy or CN6 alkoxy; 23 201106986 R2 is a hydroxyl group or a Q.6 alkoxy group; but & R2 is not a hydroxyl group at the same time. 6. The composition according to item 5 of the patent application' wherein the compound is 7. A composition according to claim 5, which has whitening and antioxidant properties. 8. A composition according to claim 7 of the patent application, which is applicable to whitening or anti-aging individual skin. 9. The composition according to claim 8 of the patent application, wherein the system is a mammal. 10. The composition according to item 7 of the patent application's can be used as a raw material for cosmetics, skin care products or health foods. 11. The composition according to item 7 of the patent application, which has the ability to inhibit melanin production. 12. A composition according to claim 11 of the invention, which inhibits the protein of the small eye-shaped transcription factor (MITF), the citase or the glycine-related protein-2 (TRP-2). which performed. 13. The composition according to item 7 of the patent application, which has the ability to scavenge free radicals. 14. The composition of claim 13 wherein the free radical is a DppH free radical and an ABTS free radical. 15. The composition according to claim 7 of the patent application's which reduces the content of intracellular reactive oxygen species (R〇ss). 16. The composition according to item 7 of the patent application, which has the ability to inhibit lipid peroxidation. twenty four